Positron Emission Tomography (PET) is a molecular imaging modality that utilizes radiolabeled tracer molecules to target, image and quantify biological processes in vivo. PET tracers can be used to study disease mechanisms, to develop novel diagnostics and therapeutics, detect early stage disease, and monitor response to therapy. Due to low density of receptors in the CNS (e.g. picomole/gram tissue receptor concentration), it is critical to synthesize PET radioligands with high specific activity (SA) for n vivo neuroimaging. In particular, very high SA is needed to obtain good image contrast in brains of small animals to ensure that receptor occupancy remains at tracer levels (<5%) and that pharmacologic effects are avoided. The lack of wide availability of diverse radioligands with high SA remains the bottleneck in accelerating the translation of new radiotracers into brain imaging diagnostics or routine tools for the study of CNS disorders and treatments. Production of PET probes in general requires very expensive equipment, safety infrastructure, and highly-trained personnel. High SA production using conventional approaches increases costs and hazards even further as generally one must use very high amounts of radioactivity (>1 Ci) to start the synthesis. Performing the synthesis of [18F]Fallypride and [18F]FLT in microliter volumes in a microfluidic chip has been shown to routinely result in very high SA (20 Ci/mol or above) using low starting radioactivity (~10-20 mCi). Based on follow-up studies conducted to understand and optimize this phenomenon, the overall goal of this proposal is to construct a robust, automated, and user-friendly system based on microfluidic synthesis that would enable production of very high SA neuroimaging tracers on-site (i.e., in the imaging center). In Aim 1, an automated approach for the concentration of [18F]fluoride into a fixed volume, and delivery into the microfluidic chip, is developed. This will enable a variety of sources (i.e. different volumes and concentrations) of [18F]fluoride to be connected to the microfluidic system. In Aim 2, the remainder of the automation to complete the synthesis protocol, including purification and formulation in saline for injection, is developed. Using [18F]Fallypride as an example, the system performance will be validated and compared to results of manual production. Finally, in Aim 3, microscale synthesis protocols for three additional example CNS tracers ([18F]FET, [18F]FDDNP, and [18F]FMZ) with be developed and optimized, to demonstrate the versatility of the automated system, and to study the effect of SA on imaging in mice and rats to create guidance for the selection of optimal SA for such studies in preclinical research. It is expected that the prototype synthesis system will be applicable to a wide range of tracers, and will be scalable to the production of clinical doses. This proposal will result in a fully-automated prototype system for routine high specific activity PET tracer production that will enable imaging centers to produce several high SA PET tracers on-demand, thereby accelerating their ability to develop and translate new diagnostics and therapies for CNS disorders.